以减毒沙门氏菌携带HPV16 L1和siRNA-E6共表达质粒防治子宫颈癌的研究
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摘要
子宫颈癌是妇女最常见的恶性肿瘤之一,全球每年约有50万新诊断病例,每年约20万人死于子宫颈癌。子宫颈癌的治疗主要以手术和放疗为主、化疗为辅,但可产生严重的不良反应。寻找防治子宫颈癌的新方法仍是重要而迫切的课题。
流行病学研究发现,人乳头瘤病毒(HPV)与子宫颈癌的发生密切相关,99.7%的子宫颈癌组织中可检测到HPV,其中57.4%为HPV l6。HPV的L1衣壳蛋白是病毒的结构蛋白,具有较强的免疫原性,E6和E7蛋白是癌蛋白,与宿主细胞的恶性转化有关。因此,针对HPV制备疫苗对于防治子宫颈癌具有重要意义。
目前,虽然Merck和GlaxoSmithKline两大公司已发展了两种HPV疫苗,但是对发病后的患者无效,只能用于早期或未发病人群的预防性接种;而且该类疫苗必须使用佐剂,价格昂贵。
本课题着手于发展预防兼早期治疗性HPV疫苗,并以减毒沙门氏菌为运载体,以期实现通过局部粘膜途径对子宫颈癌进行防治的目的。(1)构建pET28a-HPV16-L1质粒,在大肠杆菌BL21(DE3)中经IPTG诱导表达HPV16-L1蛋白,免疫小鼠后在小鼠血清中检测到抗HPV16-L1抗体。(2)构建pcDNA3.1-HPV16-L1质粒,电转化至减毒沙门氏菌PhoP/PhoQ株和Ty21a中,滴鼻免疫小鼠,在小鼠血清及阴道冲洗液中检测到抗HPV16-L1抗体,且小鼠血清中IL-2和IFN-γ水平升高。(3)构建pGC-siRNA-E6和pGC-siRNA-E7质粒,转染至人子宫颈癌细胞系Siha细胞,发现HPV16 E6和E7的转录与表达水平降低,细胞出现凋亡,凋亡机制与激活线粒体凋亡通路有关。建立子宫颈癌裸鼠皮下移植瘤模型,电转染pGC-siRNA-E6质粒后,肿瘤生长受到抑制,其基因变化与体外实验一致。(4)构建pcDNA3.1-HPV16-L1-U6-siRNA-E6质粒,电转化至减毒沙门氏菌PhoP/PhoQ株,滴鼻接种于子宫颈癌裸鼠皮下移植瘤模型,结果显示HPV16-L1与siRNA-E6具有协同抑瘤作用,亦说明通过粘膜接种途径可达到对异位靶部位的生物学效应。
本文在国内外首次构建了HPV16-L1和siRNA-E6共表达质粒,并以减毒沙门氏菌为运载体,用于子宫颈癌的防治,为发展新型防治子宫颈癌疫苗提供了实验依据。
Cervical cancer is one of the most globally common cancers in women, just second to breast cancer in morbidity. Worldwide, 27 million patients each year die from cervical cancer. Currently, surgery, radiotherapy and chemotherapy still be dominant in cervical cancer treatments, but these therapys also show serious untoward reaction in patients. Study on biologic agent will be imminent in preventing and treating cervical cancer.
Epidemiological and virological data show that human papillomavirus(HPV) infection is the major etiological factor of cervical cancer. HPV DNA can be found within 99.7% of the malignant cells of cervical cancers, of which, 57.4% was HPV l6 type. The discovery indicate that cervical cancer vaccines is expected to be the first human cancer vaccines.
HPV is a small, nonenveloped virus. The genome consisit a single copy of circular double-stranded DNA including early region (E), late region (L) and upstream regulatory region (URR). E6 and E7 protein of High-risk HPV is viral oncoprotein which can lead to immortalization by interruptting the cell growth-regulatory pathways. L1 major capsid protein of HPV can self-assemble into VLPs (virus-like particles) and show good immunogenicity.
The current HPV prophylactic vaccine candidates are based on VLPs. HPV-L1 VLPs are morphologically and antigenically similar to authentic virions without infectious and carcinogenic. However, the VLPs are mainly obtained from insect cells infected by recombinant baculovirus vector or yeast with recombinant vector. The process is cumbersome, and expensive for VLPs application in developing countries.
Currently, most of therapeutic HPV vaccines optimize HPV E6 and E7 target antigen to induce cellular immune response and inhibit cancer growth. However, they are potential carcinogens, may lead to cell abnormal proliferation and other adverse reactions. RNA interference is a new gene therapy technology. Some researchers have succeed in silencing HPV-E6 and/or E7 gene of cervical cancer by RNA interference. However, siRNA molecules enter target cells and maintain stability limit its application in vivo.
Attenuated Salmonella is the first recombinant bacteria used to delivery antigen for 25 years. More and more evidence support that alive attenuated Salmonella can induce humoral and cellular immune responses in vivo. Attenuated Salmonella can induce mucosal immune response by non-injection at less than 3 times. In tumor tissue, the attenuated Salmonella can make best use of the hypoxic microenvironment of tumor, nutrients and local immunosuppressive microenvironment. It is the biology advantage of the attenuated Salmonella that make it to be a vaccine vector used for the cervical cancer prevention and therapy.
The purpose of this study is to construct pcDNA3.1-HPV16-L1-siRNA-E6 plasmid, transform it into attenuated Salmonella, vaccinate mice by intranasal, and observe the prevention and treatment effect of cervical cancer.
The result proved, attenuated Salmonella carrying pcDNA3.1-HPV16-L1 induced anti-HPV16-L1 antibodies in mice successfully after vaccinate intranasally. siRNA-E6 designed by the sequence of HPV16-E6E7, and plasmid carrier attenuated Salmonella was innovative applicated in the cervical cancer treatment and result in significant antitumor effect. The recombinant attenuated Salmonella with pcDNA3.1-HPV16-L1-siRNA-E6 plasmid also success contribute to cervical cancer prevention and treatment.
1. Attenuated Salmonella carrying pcDNA3.1-HPV16-L1 as DNA vaccine
As the purification process of recombinant protein is complicated, protein vaccine is expensive. Further more, the multivalent protein vaccine is available. One of main purpose of this study is to develop preventive DNA vaccine. In this study, HPV16-L1 gene was amplified from fresh human cervical cancer genom, prokaryotic expression recombinant plasmid pET28a-HPV16-L1 was constructed and transfered into E. coli BL21(DE3) to express HPV16-L1 protein.
After denatured and purified, virus-like particles of 50nm diameter could be observed by transmission electron microscope. Anti-HPV16-L1 antibody could be detected in the serum fluid of mice, after mice be vaccined with emulsified VLPs and Freund adjuvant.
Further more, the eukaryotic expression plasmid pcDNA3.1-HPV16-L1 was construced and transfected into hamster kidney cell line BHK with liposome. After 48h, target protein was detected. The pcDNA3.1-HPV16-L1 was transformed into attenuated Salmonella Ty21a and PhoP/PhoQ used to vaccined model mice by intranasal immunization. Anti-HPV16-L1 antibodies can be found in serum and vaginal fluid of mice. The antibody levels in serum and vaginal fluid was increased with time, reached a peak in the 15th day and maintain a high level to the end of the experiment. In this study, the serum content of IL-2 and INF-γincreased in the group of attenuated Salmonella carrying pcDNA3.1-HPV16-L1, compared with the control group.
2. siRNA-HPV16-E6 inhibit the proliferation of cervical cancer
At present, most of therapeutic HPV vaccine induce cellular immune response by HPV E6 and E7 proteins. E6 and E7 proteins may lead to cell abnormal proliferation for its potential carcinogens. In this study, siRNAs were emploied to silence HPV E6 and E7 oncogene and inhibit the growth of cervical cancer.
HPV E6 and E7 gene sequences was used to design siRNA-E6A, siRNA-E6B and siRNA-E7 for constructing pGC-siRNA plasmid. After transfected into cervical cancer Siha cells, pGC-siRNA in siE6A group and siE7 group down-regulat the E6 and E7 transcription and expression, inhibit cell growth, promote cell apoptosis, and up-regulate the p53, bax, caspase-9 and caspase-3 expression.
After the cervical cancer model mice was established, pGCsi-E6A plasmid was transfected into tumor tissue. The E6 and E7 expression were dowm-regulated, tumor growth was inhibited, the apoptosis-related genes expression were up-regulated in the experimental group compared with control group. All datum support pGCsi-E6A plasmid has antitumor effect in vivo.
3. Cervical cancer prevention and therapy by attenuated Salmonella carrying pcDNA3.1-HPV16-L1-siRNA-E6
In this study, pcDNA3.1-HPV16-L1-siRNA-E6 was constructed for the first time, transformed into attenuated Salmonella PhoP/PhoQ to observe the treatment effect of tumor model mice. RT-PCR, western blotting and immunohistochemistry assay showed that L1 expression is upregulated in tumor tissues, whereas E6 and E7 expression are downregulated. The results indicating that attenuated Salmonella could carry the plasmid to tumor tissue and realize HPV16-L1 protein expression and E6E7 gene silenc successfully. Attenuated Salmonella with empty vector, pGC-siE6, pcDNA3.1-HPV16-L1, and pcDNA3.1-HPV16-L1-siRNA-E6 can inhibit tumor growth compared with the control group, suggesting that attenuated Salmonella has antitumor effect. Compared with pcDNA3.1-HPV16-L1 and pGC-siE6, co-expression plasmid can further reduce tumor size, suggesting that HPV16-L1 expression and E6E7 silencing in tumor have synergistic effect by immune regulation and RNA interference in cervical cancer.prevention and treatment.
For the cervical cancer prevention and therapy, this study will contribute to a new productive thinking.
流行病学研究发现,人乳头瘤病毒(HPV)与子宫颈癌的发生密切相关,99.7%的子宫颈癌组织中可检测到HPV,其中57.4%为HPV l6。HPV的L1衣壳蛋白是病毒的结构蛋白,具有较强的免疫原性,E6和E7蛋白是癌蛋白,与宿主细胞的恶性转化有关。因此,针对HPV制备疫苗对于防治子宫颈癌具有重要意义。
目前,虽然Merck和GlaxoSmithKline两大公司已发展了两种HPV疫苗,但是对发病后的患者无效,只能用于早期或未发病人群的预防性接种;而且该类疫苗必须使用佐剂,价格昂贵。
本课题着手于发展预防兼早期治疗性HPV疫苗,并以减毒沙门氏菌为运载体,以期实现通过局部粘膜途径对子宫颈癌进行防治的目的。(1)构建pET28a-HPV16-L1质粒,在大肠杆菌BL21(DE3)中经IPTG诱导表达HPV16-L1蛋白,免疫小鼠后在小鼠血清中检测到抗HPV16-L1抗体。(2)构建pcDNA3.1-HPV16-L1质粒,电转化至减毒沙门氏菌PhoP/PhoQ株和Ty21a中,滴鼻免疫小鼠,在小鼠血清及阴道冲洗液中检测到抗HPV16-L1抗体,且小鼠血清中IL-2和IFN-γ水平升高。(3)构建pGC-siRNA-E6和pGC-siRNA-E7质粒,转染至人子宫颈癌细胞系Siha细胞,发现HPV16 E6和E7的转录与表达水平降低,细胞出现凋亡,凋亡机制与激活线粒体凋亡通路有关。建立子宫颈癌裸鼠皮下移植瘤模型,电转染pGC-siRNA-E6质粒后,肿瘤生长受到抑制,其基因变化与体外实验一致。(4)构建pcDNA3.1-HPV16-L1-U6-siRNA-E6质粒,电转化至减毒沙门氏菌PhoP/PhoQ株,滴鼻接种于子宫颈癌裸鼠皮下移植瘤模型,结果显示HPV16-L1与siRNA-E6具有协同抑瘤作用,亦说明通过粘膜接种途径可达到对异位靶部位的生物学效应。
本文在国内外首次构建了HPV16-L1和siRNA-E6共表达质粒,并以减毒沙门氏菌为运载体,用于子宫颈癌的防治,为发展新型防治子宫颈癌疫苗提供了实验依据。
Cervical cancer is one of the most globally common cancers in women, just second to breast cancer in morbidity. Worldwide, 27 million patients each year die from cervical cancer. Currently, surgery, radiotherapy and chemotherapy still be dominant in cervical cancer treatments, but these therapys also show serious untoward reaction in patients. Study on biologic agent will be imminent in preventing and treating cervical cancer.
Epidemiological and virological data show that human papillomavirus(HPV) infection is the major etiological factor of cervical cancer. HPV DNA can be found within 99.7% of the malignant cells of cervical cancers, of which, 57.4% was HPV l6 type. The discovery indicate that cervical cancer vaccines is expected to be the first human cancer vaccines.
HPV is a small, nonenveloped virus. The genome consisit a single copy of circular double-stranded DNA including early region (E), late region (L) and upstream regulatory region (URR). E6 and E7 protein of High-risk HPV is viral oncoprotein which can lead to immortalization by interruptting the cell growth-regulatory pathways. L1 major capsid protein of HPV can self-assemble into VLPs (virus-like particles) and show good immunogenicity.
The current HPV prophylactic vaccine candidates are based on VLPs. HPV-L1 VLPs are morphologically and antigenically similar to authentic virions without infectious and carcinogenic. However, the VLPs are mainly obtained from insect cells infected by recombinant baculovirus vector or yeast with recombinant vector. The process is cumbersome, and expensive for VLPs application in developing countries.
Currently, most of therapeutic HPV vaccines optimize HPV E6 and E7 target antigen to induce cellular immune response and inhibit cancer growth. However, they are potential carcinogens, may lead to cell abnormal proliferation and other adverse reactions. RNA interference is a new gene therapy technology. Some researchers have succeed in silencing HPV-E6 and/or E7 gene of cervical cancer by RNA interference. However, siRNA molecules enter target cells and maintain stability limit its application in vivo.
Attenuated Salmonella is the first recombinant bacteria used to delivery antigen for 25 years. More and more evidence support that alive attenuated Salmonella can induce humoral and cellular immune responses in vivo. Attenuated Salmonella can induce mucosal immune response by non-injection at less than 3 times. In tumor tissue, the attenuated Salmonella can make best use of the hypoxic microenvironment of tumor, nutrients and local immunosuppressive microenvironment. It is the biology advantage of the attenuated Salmonella that make it to be a vaccine vector used for the cervical cancer prevention and therapy.
The purpose of this study is to construct pcDNA3.1-HPV16-L1-siRNA-E6 plasmid, transform it into attenuated Salmonella, vaccinate mice by intranasal, and observe the prevention and treatment effect of cervical cancer.
The result proved, attenuated Salmonella carrying pcDNA3.1-HPV16-L1 induced anti-HPV16-L1 antibodies in mice successfully after vaccinate intranasally. siRNA-E6 designed by the sequence of HPV16-E6E7, and plasmid carrier attenuated Salmonella was innovative applicated in the cervical cancer treatment and result in significant antitumor effect. The recombinant attenuated Salmonella with pcDNA3.1-HPV16-L1-siRNA-E6 plasmid also success contribute to cervical cancer prevention and treatment.
1. Attenuated Salmonella carrying pcDNA3.1-HPV16-L1 as DNA vaccine
As the purification process of recombinant protein is complicated, protein vaccine is expensive. Further more, the multivalent protein vaccine is available. One of main purpose of this study is to develop preventive DNA vaccine. In this study, HPV16-L1 gene was amplified from fresh human cervical cancer genom, prokaryotic expression recombinant plasmid pET28a-HPV16-L1 was constructed and transfered into E. coli BL21(DE3) to express HPV16-L1 protein.
After denatured and purified, virus-like particles of 50nm diameter could be observed by transmission electron microscope. Anti-HPV16-L1 antibody could be detected in the serum fluid of mice, after mice be vaccined with emulsified VLPs and Freund adjuvant.
Further more, the eukaryotic expression plasmid pcDNA3.1-HPV16-L1 was construced and transfected into hamster kidney cell line BHK with liposome. After 48h, target protein was detected. The pcDNA3.1-HPV16-L1 was transformed into attenuated Salmonella Ty21a and PhoP/PhoQ used to vaccined model mice by intranasal immunization. Anti-HPV16-L1 antibodies can be found in serum and vaginal fluid of mice. The antibody levels in serum and vaginal fluid was increased with time, reached a peak in the 15th day and maintain a high level to the end of the experiment. In this study, the serum content of IL-2 and INF-γincreased in the group of attenuated Salmonella carrying pcDNA3.1-HPV16-L1, compared with the control group.
2. siRNA-HPV16-E6 inhibit the proliferation of cervical cancer
At present, most of therapeutic HPV vaccine induce cellular immune response by HPV E6 and E7 proteins. E6 and E7 proteins may lead to cell abnormal proliferation for its potential carcinogens. In this study, siRNAs were emploied to silence HPV E6 and E7 oncogene and inhibit the growth of cervical cancer.
HPV E6 and E7 gene sequences was used to design siRNA-E6A, siRNA-E6B and siRNA-E7 for constructing pGC-siRNA plasmid. After transfected into cervical cancer Siha cells, pGC-siRNA in siE6A group and siE7 group down-regulat the E6 and E7 transcription and expression, inhibit cell growth, promote cell apoptosis, and up-regulate the p53, bax, caspase-9 and caspase-3 expression.
After the cervical cancer model mice was established, pGCsi-E6A plasmid was transfected into tumor tissue. The E6 and E7 expression were dowm-regulated, tumor growth was inhibited, the apoptosis-related genes expression were up-regulated in the experimental group compared with control group. All datum support pGCsi-E6A plasmid has antitumor effect in vivo.
3. Cervical cancer prevention and therapy by attenuated Salmonella carrying pcDNA3.1-HPV16-L1-siRNA-E6
In this study, pcDNA3.1-HPV16-L1-siRNA-E6 was constructed for the first time, transformed into attenuated Salmonella PhoP/PhoQ to observe the treatment effect of tumor model mice. RT-PCR, western blotting and immunohistochemistry assay showed that L1 expression is upregulated in tumor tissues, whereas E6 and E7 expression are downregulated. The results indicating that attenuated Salmonella could carry the plasmid to tumor tissue and realize HPV16-L1 protein expression and E6E7 gene silenc successfully. Attenuated Salmonella with empty vector, pGC-siE6, pcDNA3.1-HPV16-L1, and pcDNA3.1-HPV16-L1-siRNA-E6 can inhibit tumor growth compared with the control group, suggesting that attenuated Salmonella has antitumor effect. Compared with pcDNA3.1-HPV16-L1 and pGC-siE6, co-expression plasmid can further reduce tumor size, suggesting that HPV16-L1 expression and E6E7 silencing in tumor have synergistic effect by immune regulation and RNA interference in cervical cancer.prevention and treatment.
For the cervical cancer prevention and therapy, this study will contribute to a new productive thinking.
引文
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